The 5'->3' exoribonucleases (XRNs) comprise a large family of conserved enzymes in eukaryotes with important functions in RNA metabolism and RNA interference. In fungi and animals, XRN1 (175 kD) is primarily cytosolic and is involved in degradation of decapped mRNAs, nonsense mediated decay, microRNA decay and is essential for proper development. XRN2 (115 kD) is primarily nuclear, and its ortholog in S. cerevisiae, more commonly known as Rat1, has roles in RNA trafficking from the nucleus to the cytoplasm and transcription of some genes by RNA polymerase III. More recent studies have identified XRN2/Rat1 as the exoribonuclease that is essential for the 'torpedo' model of transcription termination by RNA polymerase II (Pol II). In S. cerevisiae, Rat1 co-purifies with another protein, Rai1 (Rat1 interacting protein 1), which appears to enhance the exoribonuclease activity of Rat1 in vitro. A weak sequence homolog of Rai1, known as DOM3Z, exists in mammals, although it probably does not associate with XRN2. The important functions of the XRNs in RNA metabolism, RNA interference and Pol II termination have generated significant interest in them. However, the molecular mechanism for the functions of these enzymes is currently still poorly understood. As a first step to fill these gaps in our knowledge, we recently determined the crystal structures of the Rat1-Rai1 complex as well as of Rai1 and DOM3Z alone, and carried out preliminary biochemical and functional studies based on the structural information. These results set the stage for further structural, biochemical and biological studies on these proteins. Most interestingly, our initial studies show that Rai1/DOM3Z contains a separate active site, with RNA 5' pyrophosphohydrolase activity, and we hypothesize that Rai1/DOM3Z may have important functions in RNA metabolism or quality control. We will also test this hypothesis in our proposed research project, which will help to define the physiological functions of this novel eukaryotic enzyme. PUBLIC HEALTH RELEVANCE: The proteins being studied in this proposal have not been linked to human diseases based on current information.